Integration of therapeutic cargo into the human genome with programmable type V-K CAST

CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.

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Publication Details

Journal
Nature Communications
Published
2025-03-13
DOI
https://doi.org/10.1038/s41467-025-57416-2
Citations
8
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
6.45
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article

Integration of therapeutic cargo into the human genome with programmable type V-K CAST

Christopher T. Brown, Daniela S. Aliaga Goltsman, Jennifer Oki, Audra E. Devoto et al.
8 citations
Nature Communications
CRISPR and Genetic Engineering
6.45
article

Integration of therapeutic cargo into the human genome with programmable type V-K CAST

Christopher T. Brown, Daniela S. Aliaga Goltsman, Jennifer Oki, Audra E. Devoto, Liliana Gonzalez‐Osorio, Christine A. Romano, Brian C. Thomas, Rodrigo Fregoso Ocampo, Sarah M. Laperriere, Cristina N. Butterfield, Morayma M. Temoche-Diaz, Keirstinne Turcios, Shailaja Chadha, Cindy J. Castelle, Owen P. Janson, Jahan Rahman, Lisa Alexander, Jennifer Hong, Gregory J. Cost, Drew T. Dunham, Jason Liu, Jared Muysson, Khak Khak Khayi
article en
8 citations

Abstract

CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.

Nature CommunicationsVol. 16(1)
Institute of Metagenomics and Microbial Technologies (Slovenia) (SI)
Openalex Percentile: Top 3%
CRISPR and Genetic Engineering
6.45
DOIOpen Access PDFOpenAlex
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